1. Field of the Invention
The present invention relates to the regulation of mammalian gene expression.
2. Description of Related Art
Gene transfer involves the transfer of foreign genetic material into a cell such that the foreign material is expressed. This process is used in applications such as, for example: gene therapy, production of recombinant biologicals, genetic diagnosis, and drug screening. But despite recent reports of success in the most challenging of these fields, in vivo gene therapy of human diseases (Kay et al., 2000; Cavazzano-Calvo et al., 2000), the construction of new expression vectors has occupied the attention of the many workers eager to achieve high levels of gene expression in a regulated manner (reviewed in Agha-Mohammadi and Lotze, 2000).
In most cases, the ultimate goal of gene transfer is to introduce an expression vector that provides for production of a gene product for a period sufficient for a therapeutic or prophylactic effect, which period may be relatively short (e.g., a few hours to a few days) or may be for long periods (e.g., several weeks to one or more years). One important aspect of gene-based therapy could involve regulating expression in such a manner that gene expression is restricted spatially and temporally to cells or tissues that are affected by a disease. Such regulation requires that the gene be delivered to the target cell or tissue in a substantially latent state, so that it does not change or significantly affect the phenotype of the target in the absence of disease. Where and when the disease is active, it would be desirable that the latent gene should then be induced (e.g., spatially, temporally, or both) in a manner that will counteract disease symptoms and, conversely, ceases expression as the disease symptoms subside. To simplify, this requires that the gene be regulated by a tight on/off switch that can respond to an intrinsic disease-related stimulus.
A critical feature of such regulated gene expression is called the silencer-inducer ratio: expression of the foreign gene measured under inducing conditions divided by the amount of expression without induction (i.e., basal expression). This ratio should be high (e.g., at least about 25- to 1000-fold) and sufficiently regulatable by appropriate control of inducing conditions. Another critical feature is substantially silenced (or repressed) gene expression in the non-induced, disease-free state.
This requirement for a tight on/off switch in regulating expression of a foreign gene is widely acknowledged and the absence of such regulation is considered to be one of the major limitations for many gene transfer applications. Regulated expression of foreign genes, both positive and negative, has been described in prokaryotes (e.g., the Lac operon) and in mammals (e.g., Tet-repressor and activator, progesterone or ecdysone receptor) (reviewed in Agha-Mohammadi and Lotze, 2000). Each of these systems involves binding of an extrinsic modulator to a protein involved in transcription: tetracycline or doxycycline in the Tet regulatory system; RU486 or rapamycin in the progesterone and FKBP regulatory systems, respectively. The latter two systems require multiple vectors to deliver the target gene and the different regulatory components. In all of these systems, allosteric changes determine the DNA binding affinities of positive- and negative-acting transcriptional factors and thereby control an on/off switch (Freundlieb et al., 1999). Unlike the invention, however, these systems do not provide spatial regulation within a tissue or responsiveness to a disease state by an intrinsic factor (e.g., hypoxia or stress) acting on endogenous transcriptional factors (e.g., hypoxia inducible factors or NF-κB transcription factors, respectively). A system of regulated expression has been engineered in yeast where allosteric activation (i.e., phosphorylation) of positive- or negative-acting factors activate or repress transcription (Lee and Gross, 1993). These systems provide a solution to the problem of providing a tight on/off switch for regulated expression by using allosteric binding and an extrinsic modulator to control activity of a promoter. As compared to the invention described herein, these systems are all dissimilar in mechanism because this invention uses disease-responsive intrinsic factors to mediate spatial as well as temporal reversible repression, but does not depend upon allosteric binding. Therefore, allosteric regulatory systems do not teach or suggest the invention.